BDNF Mediates the Effects of Testosterone on the Survival of New Neurons in an Adult Brain  S Rasika, Arturo Alvarez-Buylla, Fernando Nottebohm  Neuron  Volume 22, Issue 1, Pages 53-62 (January 1999) DOI: 10.1016/S0896-6273(00)80678-9

Figure 1 TrkB and BDNF Localization in Male and Female HVC TrkB immunoreactive cells and neuropil are abundant in the HVC of both males and females, with only a few weakly stained immunopositive cells scattered outside the HVC. Cells that are weakly immunopositive to BDNF are present in the HVC of adult males perfused conventionally (i.e., with saline followed by paraformaldehyde). Immunostaining is dramatically increased after perfusion with acetate-buffered saline (ABS [pH 3.0]) and paraformaldehyde, although cellular morphology is lost. In contrast, very little BDNF immunopositivity can be seen in the HVC of adult females, regardless of perfusion method. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)

Figure 2 Diagram of Cannula Placement and Schematic of Protocols Used for the Infusion Experiments (A) Protocol for the first infusion experiment. Two groups of female canaries were treated twice daily with injections of 3H-thymidine for 5 days. Twenty-five days after the first injection, carrier (saline+BSA) or BDNF was infused for 10 days and the animals were killed. (B) Protocol for the second infusion experiment. Three groups of female canaries were treated with injections of 3H-thymidine twice daily for 5 days. Twenty-five days after the first injection, the first group received empty Silastic implants and infusion of carrier (saline+BSA), the second group received testosterone-filled Silastic implants and infusion of carrier, while the third group received testosterone-filled Silastic implants and infusion of α-BDNF into the HVC. Animals were killed 10 days after infusion began. (C) Coronal section through the HVC, and the point of insertion of the cannula, with the bevel facing the HVC. The extent of the paraffin coating on the osmotic pump is also indicated. (D and E) Representative parasagittal sections at the levels d′ and e′, respectively, shown in (C), to indicate positions of HVC and control areas mapped after BDNF infusion. Caudal HV—caudal part of hyperstriatum ventrale; LFM—lamina frontalis suprema; LH—lamina hyperstriatica; LMD—lamina medullaris dorsalis; NCM—caudomedial neostriatum; V—lateral ventricle. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)

Figure 3 Photomicrograph of 3H-Labeled and Unlabeled HVC Neurons in a Section Counterstained with Cresyl Violet Note the 3H-labeled neuron in the center with a large clear nucleus and the presence of numerous exposed silver grains that resulted from autoradiography of 3H-thymidine. Unlabeled neurons in focus are indicated with black arrows. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)

Figure 4 Effect of BDNF on the Total Number of New (3H-Labeled) Neurons in the HVC and HVC Volume An asterisk (*) indicates a significant effect of BDNF infusion (ANOVA). The total number of new or 3H-labeled neurons triples with BDNF treatment, while the volume of the HVC increases about 60%, in both seasons. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)

Figure 5 Effect of BDNF Infusion on the Density of New (3H-Labeled) Neurons in the HVC and Three Control Regions An asterisk (*) indicates a significant effect of BDNF infusion. BDNF infusion significantly increases the density of new neurons in the HVC in both seasons but does not change the density of new neurons in the control regions: the shelf under the HVC, caudal HV (hyperstriatum ventrale), and NCM (caudomedial neostriatum), in either spring or fall. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)

Figure 6 Effect of Testosterone Treatment on BDNF Levels in the HVC Both the size of the HVC and immunostaining for BDNF are increased in testosterone-treated (T-treated) females when compared to controls, as revealed by perfusion with acetate-buffered saline (ABS [pH 3.0]) and paraformaldehyde. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)

Figure 7 Effect of Hormonal Manipulation and Infusion of α-BDNF on the Total Number of New (3H-labeled) Neurons in the HVC and HVC Volume An asterisk (*) indicates a significant effect of treatment (ANOVA). Testosterone treatment alone results in the doubling of the total number of new or 3H-labeled neurons and an 18% increase in the volume of the HVC, when compared to control females. However, α-BDNF infusion neutralizes this effect of testosterone treatment, retaining both the number of new neurons and HVC volume at control levels. Neuron 1999 22, 53-62DOI: (10.1016/S0896-6273(00)80678-9)